The preparation of polypeptides using recombinant technology has developed into a standard procedure during the last couple of decades. The access to recombinant polypeptides by cloning the genes encoding the respective polypeptide followed by subsequent transformation of suitable expression hosts with the gene to be expressed and final production and purification of the obtained recombinant polypeptide product has provided access to a whole new class of biologically designed and produced therapeutics.
Pharmaceutically active compounds have been prepared in increasing numbers in the pharmaceutical industry using recombinant DNA technology followed by production processes developed in the field of bioengineering.
Such biological products include monoclonal antibodies, which have been developed into important treatment options in various medical fields including autoimmune diseases, inflammatory disorders, immunosuppression, oncology or the like.
Development of such therapeutics of biological origin requires production at industrial scale thereby providing access to large amounts of recombinant polypeptide. Preferred expression systems are mammalian cell cultures which are superior to most other eukaryotic systems based on insect cells, yeast or the like, or even traditional prokaryotic expression systems.
However, mammalian cell culture includes tremendous challenges especially at the industrial scale. Production facilities for mammalian cell culture require thorough optimization of many process conditions.
In particular, cell culture processes for the production of polypeptides in mammalian cells require continuous optimization of the culture conditions and their adaptation to specific cell lines or products in order to reach a high volumetric product yield in combination with optimal product quality.
Much previous effort has concentrated on the basic parameters of cell culture media including their composition concerning e.g., kinds and concentrations of ions, amino acids, vitamins or trace elements or the osmolality of the medium. Further important parameters, which have been in the focus of research, are e.g. feed composition or feeding schedules to reach optimal cell growth.
Also temperature and pH as basic physiological parameters are known to have significant influence on the culturing of mammalian cells. The temperature in general considerably affects the growth state and the viability of cells. In addition to this, it may, however, also more specifically influence the polypeptide product and its characteristics by altering e.g. the glycosylation (US 2003/0190710 A1; EP 1 373 547 A1; US 2004/0214289 A1).
The pH at which the growth medium and the cells are maintained can also influence and alter cell growth and polypeptide production in a specific manner that depends on the particular cell line and product (Sauer et al. Biotechnology and Bioengineering 2000, Vol 67, pg. 586-597: Yoon et al., Biotechnology and Bioengineering 2004, Vol 89, pg. 346-356; Kuwae et al., Journal of Bioscience and Bioengineering 2005 Vol 100, pg. 502-510).
Over the time course of culturing, the requirements of the cells may change. While in the beginning it is advantageous to optimize conditions towards improved cell growth, in later stages enhanced cell survival and maintenance of the viable cell density in connection with obtaining high product titers become important. In this respect introducing one or more temperature steps during cell culturing has been suggested (Chen et al., J Biosci Bioeng. 2004; 97 (4):239-43). For this, mammalian cells are cultured at least at two different temperatures, wherein the first higher temperature is optimized for cell growth while the second or third lower temperature is selected to improve the productivity of the cells (e.g. Weidemann et al., Cytotechnology. 1994, 15(1-3):111-6: WO 00/36092; EP 0 764 719 A2, US 2005/019859, EP 1 575 998, US 2008/081356). Other documents describe the use of temperature steps in combination with additional specific media features. EP 1 757 700 A2 e.g. discloses a temperature step in combination with the presence of butyrate salts as a media component, while EP 1 789 571 A1 describes a temperature step combined with a defined amino acid content.
Also other cell culture conditions have been changed. U.S. Pat. No. 5,856,179 has introduced a method for producing polypeptides in a fed batch cell culture, wherein during culturing the osmolality of the medium is considerably altered from around 280-330 mOsm in the major growth phase to about 400-600 mOsm during the production phase.
WO 02/101019 has looked at many specific media components such as glutamine and glucose concentrations, including changes in temperature and pH. However, it was found that a pH shift in high glucose media has a negative impact on the culture and that it is not recommended to reduce the pH during the growth or production phase.
WO 2008/026445 discloses a method for production of polypeptides wherein cell culture conditions are changed from one set of culture conditions to a second set and wherein this change is combined with specific media features concerning the content of specific amino acids. The change in conditions specifically relates to shifts in temperature. Other changes in conditions such as pH or osmolality are generally mentioned as additional options, however, particular parameter settings are not specified.
Considering the above challenges and existing disadvantages, there is a continued need in the field of industrial biotechnology for improved culture processes which allow producing recombinant polypeptides at an industrial scale with even higher yields, i.e. improved specific and overall productivity, and increased product quality.
A specific technical objective of polypeptide production processes is to maintain high cell viabilities and to maximize the final yield of polypeptide by optimizing the parameters of the overall cell cultivation process.